Project Summary Pseudomonas aeruginosa is the most common and problematic bacterial pathogen in patients with chronic lower airway diseases, including cystic fibrosis (CF), non-CF bronchiectasis and chronic obstructive pulmonary disease (COPD). While each of these diseases is characterized by chronic lung inflammation, Pseudomonas aeruginosa persists and avoids clearance by innate immune cells such as macrophages and neutrophils through an unknown mechanism. Our poor understanding of how Pseudomonas aeruginosa avoids clearance by innate immune cells is a fundamental gap in our knowledge that prevents us from developing new therapeutics that can improve innate immune cell function to help clear chronic P. aeruginosa infections. Our preliminary data show that highly abundant 4-hydroxy-2-alkyl quinolones (AQs) secreted by P. aeruginosa inhibit innate immune cell function. We find that AQs, which are present in sputum at micromolar concentrations, inhibit oxidative burst in phagocytic immune cells, and limit bacterial killing. We also find that AQs dysregulate mammalian signaling pathways that regulate oxidative burst. AQs activate the host kinase AMPK, a strong negative regulator of the host mammalian target of rapamycin (mTORC1) kinase. Intriguingly, mTORC1 activity is necessary for oxidative burst and phagocytic cell function. We hypothesize that P. aeruginosa AQs inhibit phagocytic cell function in chronic airway diseases by activating AMPK and inhibiting mTORC1, thereby preventing immune clearance of pathogens. The goals of this proposal are to: 1) determine the mechanism by which AQs activate AMPK to inhibit mTORC1 signaling and 2) determine the role of AQ- mediated mTORC1 inhibition on phagocytic cell function. Together these results shift the paradigm for how P. aeruginosa establishes and maintains persistent infection in chronic lower airway diseases. Our results suggest that secreted P. aeruginosa AQs actively inhibit the bactericidal activity of recruited phagocytic cell by altering mammalian cell signaling. Defining how AQs inactivate local innate immune cells will provide novel therapeutic targets that could improve immune cell function in chronic airway disease, leading to improved clearance of persistent bacterial infection.